Glucocorticoids serve a critical role in tolerance to stress and maintenance of normal metabolic functions. Inappropriate secretion of these hormones can lead to primary disease or contribute to the pathogenesis of others. The principal determinant of glucocorticoid secretion rate is the circulating concentration of adrenocorticotropin (ACTH), a product of the pituitary corticotroph. Net secretion of ACTH represents the interaction of positive (mainly hypothalamic peptide) and negative signals acting at the corticotroph. The goal of the proposed study is to understand the cellular mechanism whereby glucocorticoids, the primary negative regulators of ACTH secretion at the pituitary, inhibit its release. Ongoing research involving this question supports the idea that glucocorticoids cause the rapid (within minutes) induction of new mRNA and protein in corticotrophs which then acts to suppress ACTH release at a central locus, common to all pathways capable of stimulating secretion. Our preliminary data suggests that the actin cytoskeleton is the likely site for this effect The protein mediating these effects needs to be identified and characterized to ultimately understand how and where negative feedback works in corticotrophs. The major goal of this study is to identify this protein(s). Since this material functions intracellularly, a system to detect its activity will be developed using sheep pituitary cells permeabilized with a bacterial toxin, a treatment which causes the formation of holes in the cell membrane permitting added proteins access to the oxocytocis process. Cytosols from AtT-20 corticotrophic tumor cells treated with glucocorticoids will be fractionated using HPLC and tested using permeabilized pituitary cells to identify and characterize the inhibitory protein. Another objective will use subtractive hybridization to isolate genes specifically activated by glucocorticoid treatment These genes will be sequenced and possibly identified by comparison with information in gene data banks. Proteins coded by these genes will subsequently be tested in the permeabilized cell system to characterize their inhibitory action on ACTH secretion. Identification of the specific intracellular product (s) mediating early glucocorticoid negative feedback could lead to important new understandings of this fundamental biologic process.